There are two major types of cells grown in vitro; suspension cells (anchorage-independent cells); and adherent cells (anchorage-dependent cells). Suspension or anchorage-independent cells can multiply, in vitro, without being attached to a surface. In contrast, adherent cells require attachment to a surface in order to grow in vitro. Additionally, some non-adherent cells grow best on a surface that promotes adherent cell growth.
It is known to grow adherent cells, in vitro, in polystyrene flasks. Polystyrene is the most common type of plastic used in the manufacture of rigid, gas impermeable cell culture flasks or plates. It is thought that polystyrene promotes the growth of adherent cells because of its ability to maintain electrostatic charges on its surface which attract oppositely charged proteins on the cell surfaces. However, to date, the available polystyrene culture containers have been of the rigid flask or plate type because polystyrene is known in the art as a rigid, gas-impermeable plastic.
Cells are commonly cultured in a growth medium within polystyrene or other containers placed in enclosed incubators. In addition to providing a certain degree of isolation from pathogens, the incubators maintain a constant temperature, usually 37.degree. C., and a constant gas mixture. The gas mixture must be optimized for a given cell type, and be controlled for at least two parameters: (1) partial pressure of oxygen (pO.sub.2) to serve the aerobic needs of the cells, and (2) partial pressure of carbon dioxide (pCO.sub.2) to maintain the pH of the growth medium. Since the known types of rigid cell culture containers are gas impermeable, their lids or caps are not sealed onto the containers. Rather, they are offset sufficiently to allow gas exchange through a gap or vent between the cap and the container. Such a container is disadvantageous for clinical uses because the vent might allow contamination of the culture or lead to accidents involving biohazardous agents.
In addition to polystyrene flasks, others have constructed flexible, breathable containers for containing adherent cells to be grown in vitro. For example, the commonly assigned U.S. Pat. No. 4,939,151 provides a gas-permeable bag with at least one access port. This allows for a closed system (ie., one without a vent). The bag disclosed in the '151 Patent is constructed from two side walls. The first side wall is made of ethylene-vinyl acetate ("EVA") which may be positively or negatively charged. The second side wall is constructed from a gas permeable film such as ethylene-vinyl acetate or a polyolefin. The first side wall is sealed to the second side wall along their edges. While EVA can hold an electrostatic charge, the charge has the undesirable tendency to decay over time. Eventually, the decay of the charge on EVA will render the container ineffective for growing adherent cells. Rigid styrene flasks with an electrostatic charge are known, and show less of a tendency to lose charge over time.
It has been found that the cell growth rate within a sealed container may be influenced by the gas permeability characteristics of the container walls. The optimal gas requirements, however, vary by cell type and over the culture period. Thus, it is desirable to be able to adjust the gas permeability of the container. The polystyrene flask, and the flexible flask which is entirely constructed from a monofilm, do not provide for such adjustability.